Composition and method for treatment of lca10 using rna-guided nuclease

Abstract

The present invention relates to LCA10 treatment using a CRISPR/Cas12f1(Cas14a1) system. In particular, the present invention relates to a composition comprising a CRISPR/Cas12f1(Cas14a1) system for LCA10 treatment, and to a treatment method using same. In addition, the present invention relates to a composition comprising a CRISPR/Cas12f1(Cas14a1) system for artificially manipulating the CEP290 gene, and to a method of editing the CEP290 gene.

1 . A guide RNA for a CRISPR/Cas12f1 system, comprising:
 a scaffold region represented by a nucleotide sequence selected from SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 477, and SEQ ID NO: 478;   a spacer region comprising, as a guide sequence, a nucleotide sequence selected from SEQ ID NOS: 51 to 101 and SEQ ID NOS: 245 to 272; and   a U-rich tail,   wherein the scaffold region, the guide region, and the U-rich tail are sequentially linked to each other in a 5′ to 3′ direction,   the scaffold region is a region capable of interacting with a Cas12f1 protein, the guide region is capable of forming a complementary bond with a target sequence present in CEP290 gene, and   the U-rich tail is represented by a sequence selected from 5′-(UaN)dUe-3′, 5′-UaVUaVUe-3′, and 5′-UaVUaVUaVUe-3′ wherein N is A, C, G or U, each V is independently A, C or G, a is an integer from 0 to 4, d is an integer from 0 to 3, and e is an integer from 1 to 10.   
     
     
         2 . A DNA encoding the guide RNA of  claim 1 . 
     
     
         3 . A guide RNA-Cas12f1 protein complex, comprising:
 a first Cas12f1 protein represented by an amino acid sequence selected from SEQ ID NO: 129 or SEQ ID NO: 288;   a second Cas12f1 protein having the same amino acid sequence as the first Cas12f1 protein; and   a guide RNA in which a scaffold region represented by a nucleotide sequence selected from SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 477, and SEQ ID NO: 478, a spacer region comprising, as a guide sequence, a nucleotide sequence selected from SEQ ID NOS: 51 to 101 and SEQ ID NOS: 245 to 272, and a U-rich tail are sequentially linked to each other in a 5′ to 3′ direction,   wherein the first Cas12f1 protein and the second Cas12f1 protein form a dimer, the scaffold region of the guide RNA and the dimer formed by the Cas12f1 proteins interact with each other to form a complex,   the U-rich tail is represented by a sequence selected from 5′-(UaN)dUe-3′, 5′-UaVUaVUe-3′, and 5′-UaVUaVUaVUe-3′ wherein N is A, C, G or U, each V is independently A, C or G, a is an integer from 0 to 4, d is an integer from 0 to 3, and e is an integer from 1 to 10, and   the guide region is capable of forming a complementary bond with a target sequence present in CEP290 gene.   
     
     
         4 . A vector comprising:
 a nucleic acid that encodes a Cas12f1 protein represented by an amino acid sequence selected from SEQ ID NO: 129 or SEQ ID NO: 288; and   a nucleic acid that encodes a guide RNA, in which a scaffold region represented by a nucleotide sequence selected from SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 477, and SEQ ID NO: 478, a spacer region comprising, as a guide sequence, a nucleotide sequence selected from SEQ ID NOS: 51 to 101 and SEQ ID NOS: 245 to 272, and a U-rich tail are sequentially linked to each other, wherein the U-rich tail is represented by a sequence selected from 5′-(UaN)dUe-3′, 5′-UaVUaVUe-3′, and 5′-UaVUaVUaVUe-3′ wherein N is A, C, G or U, each V is independently A, C or G, a is an integer from 0 to 4, d is an integer from 0 to 3, and e is an integer from 1 to 10.   
     
     
         5 . A vector comprising:
 a nucleic acid that encodes a Cas12f1 protein represented by an amino acid sequence selected from SEQ ID NO: 129 or SEQ ID NO: 288; and   a nucleic acid that encodes a first guide RNA in which a first scaffold region represented by a nucleotide sequence selected from SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 477, and SEQ ID NO: 478, a first spacer region comprising, as a guide sequence, a nucleotide sequence selected from SEQ ID NOS: 51 to 74 and SEQ ID NOS: 245 to 256, and a first U-rich tail are sequentially linked to each other; and   a nucleic acid that encodes a second guide RNA in which a second scaffold region represented by a nucleotide sequence selected from SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 477, and SEQ ID NO: 478, a second spacer region comprising, as a guide sequence, a nucleotide sequence selected from SEQ ID NOS: 75 to 101 and SEQ ID NOS: 257 to 272, and a second U-rich tail are sequentially linked to each other, wherein the first U-rich tail and the second U-rich tail are each independently represented by a sequence selected from 5′-(UaN)dUe-3′, 5′-UaVUaVUe-3′, and 5′-UaVUaVUaVUe-3′ wherein N is A, C, G or U, each V is independently A, C or G, a is an integer from 0 to 4, d is an integer from 0 to 3, and e is an integer from 1 to 10.   
     
     
         6 . The vector of  claim 4 , further comprising at least one unit of a shRNA, which decreases activity of one or more genes selected from ATM1, XRCC4, XLF-1, XRCC6, LIG4, and DCLRE1C, or a nucleic acid encoding the shRNA. 
     
     
         7 . The vector of  claim 4 , wherein the vector is one or more selected from:
 plasmid; mRNA (transcript); PCR amplicon; retroviral (retrovirus) vector;   lentiviral (lentivirus) vector; adenoviral (adenovirus) vector; adeno-associated viral (adeno-associated virus; AAV) vector; vaccinia viral (vaccinia virus) vector; poxviral (poxvirus) vector; and herpes simplex viral (herpes simplex virus) vector.   
     
     
         8 . The vector of  claim 4 , wherein the vector is a single vector. 
     
     
         9 . The vector of  claim 6 , wherein the first spacer region of the first guide RNA comprises, as a guide sequence, the nucleotide sequence of SEQ ID NO: 64, the second spacer region of the second guide RNA comprises, as a guide sequence, the nucleotide sequence of SEQ ID NO: 92 or SEQ ID NO: 96, the vector comprises two units of the nucleic acid encoding the shRNA, and the vector is an adeno-associated viral vector and is a single vector. 
     
     
         10 . A CRISPR/Cas12f1 composition comprising:
 a Cas12f1 protein represented by an amino acid sequence selected from SEQ ID NO: 129 or SEQ ID NO: 288, or a nucleic acid encoding the Cas12f1 protein; and   a guide RNA in which a scaffold region represented by a nucleotide sequence selected from SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 477, and SEQ ID NO: 478, a spacer region comprising, as a guide sequence, a nucleotide sequence selected from SEQ ID NOS: 51 to 101 and SEQ ID NOS: 245 to 272, and a U-rich tail are sequentially linked to each other, or a nucleic acid encoding the guide RNA, wherein the Cas12f1 protein and the scaffold region of the guide RNA are capable of interacting with each other to form a complex,   the spacer region of the guide RNA is capable of forming a complementary bond with a target sequence present in CEP290 gene, and   the U-rich tail is represented by a sequence selected from 5′-(UaN)dUe-3′, 5′-UaVUaVUe-3′, and 5′-UaVUaVUaVUe-3′ wherein N is A, C, G or U, each V is independently A, C or G, a is an integer from 0 to 4, d is an integer from 0 to 3, and e is an integer from 1 to 10.   
     
     
         11 . A CRISPR/Cas12f1 composition comprising:
 a Cas12f1 protein represented by an amino acid sequence selected from SEQ ID NO: 129 or SEQ ID NO: 288, or a nucleic acid encoding the Cas12f1 protein; and   a first guide RNA in which a first scaffold region represented by a nucleotide sequence selected from SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 477, and SEQ ID NO: 478, a first spacer region comprising, as a guide sequence, a nucleotide sequence selected from SEQ ID NOS: 51 to 74 and SEQ ID NOS: 245 to 256, and a first U-rich tail are sequentially linked to each other, or a nucleic acid encoding the first guide RNA; and   a second guide RNA in which a second scaffold region represented by a nucleotide sequence selected from SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 477, and SEQ ID NO: 478, a second spacer region comprising, as a guide sequence, a nucleotide sequence selected from SEQ ID NOS: 75 to 101 and SEQ ID NOS: 257 to 272, and a second U-rich tail are sequentially linked to each other, or a nucleic acid encoding the second guide RNA,   wherein the Cas12f1 protein and the first scaffold region are capable of interacting with each other to form a complex,   the Cas12f1 protein and the second scaffold region are capable of interacting with each other to form a complex,   the first spacer region and the second spacer region are each independently capable of forming a complementary bond with a target sequence present in CEP290 gene, and   the first U-rich tail and the second U-rich tail are each independently represented by a sequence selected from 5′-(UaN)dUe-3′, 5′-UaVUaVUe-3′, and 5′-UaVUaVUaVUe-3′ wherein N is A, C, G or U, each V is independently A, C or G, a is an integer from 0 to 4, d is an integer from 0 to 3, and e is an integer from 1 to 10.   
     
     
         12 . The CRISPR/Cas12f1 composition of  claim 10 , further comprising at least one unit of a shRNA, which decreases activity of one or more genes selected from ATM1, XRCC4, XLF-1, XRCC6, LIG4, and DCLRE1C, or a nucleic acid encoding the shRNA. 
     
     
         13 . The CRISPR/Cas12f1 composition of  claim 12 , wherein the CRISPR/Cas12f1 composition comprises the first guide RNA and the second guide RNA,
 the first spacer region of the first guide RNA comprises, as a guide sequence, the nucleotide sequence of SEQ ID NO: 64,   the second spacer region of the second guide RNA comprises, as a guide sequence, the nucleotide sequence of SEQ ID NO: 92 or SEQ ID NO: 96, and   the CRISPR/Cas12f1 composition comprises two units of the shRNA or the nucleic acid encoding the shRNA.   
     
     
         14 . A pharmaceutical composition for treatment of LCA10, comprising:
 the CRISPR/Cas12f1 composition of  claim 11 ; and   a pharmaceutically acceptable carrier.   
     
     
         15 . The pharmaceutical composition of  claim 14 , further comprising a shRNA, which decreases activity of one or more genes selected from ATM1, XRCC4, XLF-1, XRCC6, LIG4, and DCLRE1C, or a nucleic acid encoding the shRNA. 
     
     
         16 . A method for editing CEP290 gene that comprises IVS26 mutation (c.2991+1655A>G), comprising:
 introducing a CRISPR/Cas12f1 composition into a subject,   wherein the CRISPR/Cas12f1 composition comprises:   a Cas12f1 protein represented by an amino acid sequence selected from SEQ ID NO: 129 or SEQ ID NO: 288, or a nucleic acid encoding the Cas12f1 protein; and   a first guide RNA in which a first scaffold region represented by a nucleotide sequence selected from SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 477, and SEQ ID NO: 478, a first spacer region comprising, as a guide sequence, a nucleotide sequence selected from SEQ ID NOS: 51 to 74 and SEQ ID NOS: 245 to 256, and a first U-rich tail are sequentially linked to each other, or a nucleic acid encoding the first guide RNA; and   a second guide RNA in which a second scaffold region represented by a nucleotide sequence selected from SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 477, and SEQ ID NO: 478, a second spacer region comprising, as a guide sequence, a nucleotide sequence selected from SEQ ID NOS: 75 to 101 and SEQ ID NOS: 257 to 272, and a second U-rich tail are sequentially linked to each other, or a nucleic acid encoding the second guide RNA,   wherein the Cas12f1 protein and the first scaffold region are capable of interacting with each other to form a complex,   the Cas12f1 protein and the second scaffold region are capable of interacting with each other to form a complex,   the first spacer region of the first guide RNA of the CRISPR/Cas12f1 composition targets a region between the 3′ end of exon 26 of the CEP290 gene and the IVS26 mutation (c.2991+1655A>G) of the CEP290 gene,   the second spacer region of the second guide RNA of the CRISPR/Cas12f1 composition targets a region between the IVS26 mutation (c.2991+1655A>G) of the CEP290 gene and the 5′ end of exon 27 of the CEP290 gene,   the first U-rich tail and the second U-rich tail are each independently represented by a sequence selected from 5′-(UaN)dUe-3′, 5′-UaVUaVUe-3′, and 5′-UaVUaVUaVUe-3′ wherein N is A, C, G or U, each V is independently A, C or G, a is an integer from 0 to 4, d is an integer from 0 to 3, and e is an integer from 1 to 10, and   introduction of the CRISPR/Cas12f1 composition to the subject induces removal of a partial nucleic acid sequence, which comprises the IVS26 mutation (c.2991+1655A>G), in a region of intron 26 of the CEP290 gene.   
     
     
         17 . The method of  claim 16 , wherein the introduction of the CRISPR/Cas12f1 composition into the subject is done by administering the CRISPR/Cas12f1 composition to a human, or a non-human animal having the CEP290 gene. 
     
     
         18 . The method of  claim 16 , wherein the introduction of the CRISPR/Cas12f1 composition into the subject is done by introducing the CRISPR/Cas12f1 composition into a human cell, a non-human animal cell having the CEP290 gene, a cell obtained from a human with LCA10 disease, or a cell obtained from a non-human animal with LCA10 disease. 
     
     
         19 . The method of  claim 16 , wherein the CRISPR/Cas12f1 composition further comprises at least one unit of a shRNA, which decreases activity of one or more genes selected from ATM1, XRCC4, XLF-1, XRCC6, LIG4, and DCLRE1C, or a nucleic acid encoding the shRNA. 
     
     
         20 . The method of  claim 19 , wherein the first spacer region of the first guide RNA of the CRISPR/Cas12f1 composition comprises, as a guide sequence, the nucleotide sequence of SEQ ID NO: 64,
 the second spacer region of the second guide RNA of the CRISPR/Cas12f1 composition comprises, as a guide sequence, the nucleotide sequence of SEQ ID NO: 92 or SEQ ID NO: 96, and   the CRISPR/Cas12f1 composition further comprises two units of the shRNA or the nucleic acid encoding the shRNA.   
     
     
         21 . A method for treating LCA10 disease, comprising:
 administering the composition of  claim 12  to a subject with LCA10 disease.   
     
     
         22 . The method of  claim 21 , wherein the subject is a human with LCA10 disease or a non-human animal with LCA10 disease. 
     
     
         23 . The method of  claim 21 , wherein the first spacer region of the first guide RNA of the CRISPR/Cas12f1 composition comprises, as a guide sequence, the nucleotide sequence of SEQ ID NO: 64,
 the second spacer region of the second guide RNA of the CRISPR/Cas12f1 composition comprises, as a guide sequence, the nucleotide sequence of SEQ ID NO: 92 or SEQ ID NO: 96, and   the composition further comprises two units of the shRNA or the nucleic acid encoding the shRNA.   
     
     
         24 - 26 . (canceled)